Effect of recombinant human troponin C on the growth of human umbilical vein endothelial cells and mouse xenograft tumor

Xianjun LIU; Huiyun ZHOU; Xianfeng DENG

ABSTRACT

BACKGROUND:

Troponin I (Tn I ) could inhibit the growth of vascular endothelial cells, inhibit neovascularization,through which to inhibit the development and metastasis of solid tumor. Similar to Tn I, TnC also exists in non-muscular tissue, but does it has the analogous activity of anticancer like Tn I ?

OBJECTIVE:

To explore the effect of recombinant human TnC (rhTnC) on the growth of human umbilical vein endothelial cells (HUV-EC) and mouse xenograft tumor.

DESIGN:

Controlled observation in vivo and in vitro.

SETTING:

Research Institute of Medicine, Chongqing K.E.W Pharmaceutical Co., Ltd. and Department of Biochemistry of Chongqing Medical University.MATERIALS The experiment was conducted in the Biochemical Laboratory of Research Institute of Medicine,Chongqing K.E.W Pharmaceutical Co., Ltd. from March 2003 to December 2004. 100 Kunming mice either male or female of 15-22 g purchased from Chongqing Academy of Chinese Materia Medica. E.coli BL21 (DE3)pLysS/pET3b-TnC provided by Chongqing K.E.W Pharmaceutical Co., Ltd. HUV-EC (Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences).

METHODS:

Human TnC cDNA was obtained from human thymus cDNA library using PCR. The colony was cloned in E.coli and a bacterial strain of gene engineering E.coli BL21 (DE3) pLysS/pET3b-TnC was obtained, which could express hTnC. The recombinant human TnC (rhTnC) was purified with affinity chromatography of Ni-NTA agarose. ①In vitro cell experiment HUV-ECs were seeded in the 96-well plates at density of 2×103 cells per well and co-cultured with rhTnC of 1, 5, 10, and 50 mg/L for 3 days. The absorption (A value) was detected with microplate reader at 540 nm and the inhibition rate of cell growth was calculated. Meanwhile, the 50% inhibiting dose (IC50 value) was assayed by LOGIT method. ②In vivo animal experiment Ascites tumor (S-180) that had been inoculated for 7-8 days was harvested. The tumor cells were diluted to 1 ×1010 L-1 and 0.2 rnL was subaxillarily and intraperitoneally injected into each mouse (50 mice in each group). The next day, the mice were randomly divided into 5 groups rhTnC 20 mg/kg group, rhTnC 10 mg/kg group, rhTnC 5 mg/kg group, Cyclophosphamide (Cy) group and control group with 10 mice in each group. The rhTnC 20,10 and 5 mg/kg groups were given administration at the corresponding doses, once a day for 7 days; 50 mg/kg Cy was given the Cy group one after an interval of day, and the same volume normal saline was given to the control group. One day after the last time of administration, all mice were killed and the tumor was harvested and weighed. The inhibition rate of tumor growth was calculated tumor inhibition rate=[(Average weight of tumor in control group-Average weight of tumor in drug group)/Average weight of control group]×100%.MAIN OUTCOME

MEASURES:

Inhibition rate of rhTnC to HUV-EC proliferation in cell experiment in vitro and mouse xenograft tumor in animal experiment in vivo.

RESULTS:

①In vitro cell culture showed that rhTnC suppressed HUV-EC proliferation in a dose-dependent manner (IC50=7.5 mg/L). ②Similar to the result of in vitro cell experiment, after intraperitoneal administration, the inhibition rate of rhTnC 5, 10, and 20 mg/kg groups was higher than that of control group (P ＜ 0.01); after subaxillary administration, the inhibition rate of rhTnC 5, 10, and 20 mg/kg groups was also higher than that of control group (P ＜ 0.05-0.01). There was no significant difference in the inhibition rate between two administration approaches (P ＞ 0.05).

CONCLUSION:

rhTnC is capable of inhibiting the proliferation of HUV-EC dose-dependently, and displays the activities of inhibiting the proliferation of HUV-EC and anti-tumor.